Stauprimide Priming of Human Embryonic Stem Cells toward Definitive Endoderm.

OBJECTIVE
In vitro production of a definitive endoderm (DE) is an important issue in stem cell-related differentiation studies and it can assist with the production of more efficient endoderm derivatives for therapeutic applications. Despite tremendous progress in DE differentiation of human embryonic stem cells (hESCs), researchers have yet to discover universal, efficient and cost-effective protocols.


MATERIALS AND METHODS
In this experimental study, we have treated hESCs with 200 nM of Stauprimide (Spd) for one day followed by activin A (50 ng/ml; A50) for the next three days (Spd-A50). In the positive control group, hESCs were treated with Wnt3a (25 ng/ml) and activin A (100 ng/ml) for the first day followed by activin A for the next three days (100 ng/ml; W/A100-A100).


RESULTS
Gene expression analysis showed up regulation of DE-specific marker genes (SOX17, FOXA2 and CXCR4) comparable to that observed in the positive control group. Expression of the other lineage specific markers did not significantly change (p<0.05). We also obtained the same gene expression results using another hESC line. The use of higher concentrations of Spd (400 and 800 nM) in the Spd-A50 protocol caused an increase in the expression SOX17 as well as a dramatic increase in mortality rate of the hESCs. A lower concentration of activin A (25 ng/ml) was not able to up regulate the DE-specific marker genes. Then, A50 was replaced by inducers of definitive endoderm; IDE1/2 (IDE1 and IDE2), two previously reported small molecule (SM) inducers of DE, in our protocol (Spd-IDE1/2). This replacement resulted in the up regulation of visceral endoderm (VE) marker (SOX7) but not DE-specific markers. Therefore, while the Spd-A50 protocol led to DE production, we have shown that IDE1/2 could not fully replace activin A in DE induction of hESCs.


CONCLUSION
These findings can assist with the design of more efficient chemically-defined protocols for DE induction of hESCs and lead to a better understanding of the different signaling networks that are involved in DE differentiation of hESCs.


Introduction
Definitive endoderm (DE) is the origin of all embryonic endodermal organs such as the thyroid, lungs, liver, pancreas and intestines (1)(2)(3)(4)(5). Endodermal organs are subject to a number of illnesses, including type 1 diabetes mellitus and liver diseases, for which cell replacement therapies are a promising cure (6)(7)(8). Human embryonic stem cells (hESCs) are considered a valuable cell source for these therapeutic applications because of their ability to self-renew and potential to differentiate into different cell types (9). Since these stem cells mimic the in vivo developmental events during differentiation, the knowledge of embryology has been used to develop different stepwise protocols to produce endodermal tissues from hESCs (10)(11)(12). The first step in these directed differentiation protocols is the induction of hESCs into DE.
Studies on amniote gastrulation show that the epiblast cells which pass through the anterior primitive streak encounter various concentrations of nodal, a member of the transforming growth factor-beta family (TGF-β) and form mesoderm, in addition to DE (13,14). Other in vitro studies indicate that WNT, phosphatidylinositol 3-kinase (PI3K) and bone morphogenic proteins (BMPs) are important signaling pathways during the DE induction of embryonic stem cells (ESCs) (10,(15)(16)(17). The main growth factor inducer in DE differentiation protocols is activin A, which is also a member of the TGF-β family and a replacement for nodal. For example, it has been shown that the use of Wnt3a and activin A induces up to 80% of hESCs to express DE-specific markers such as SOX17 (15).
During recent years, as an alternative for growth factor inducers, cell-permeable bioactive small molecules (SMs) have been introduced as a means to manipulate stem cell signaling pathways (18)(19)(20). SMs can modulate DNA, RNA and protein functions. Their modulatory functions are specific, rapid and reversible. Additionally, they are less expensive (21). SMs are able to efficiently induce ESCs into different cell fates such as neural cells (22,23), cardiomyocytes (24) and pancreatic cells (23). Inducers of definitive endoderm; IDE1/2 (IDE1 and IDE2), two SM inducers of DE formation, have the capability to efficiently produce DE cells from ESCs (25). SMs also can be used as suppressors of pluripotency in ESCs (21). For example, a 20000 SM screening study has shown that a SM named Stauprimide (Spd) can suppress pluripotency by inhibiting cellular myelocytomatosis oncogene (c-MYC) signaling. This suppression primes ESCs for lineage-specific differentiation (26).
During our previous study (27), we found that Rapamycin priming before activin A induction could efficiently differentiate hESCs into DE. We also observed high expression levels of SOX17 and FOXA2 in the hESCs which were primed with Spd before activin induction. Therefore, in this study we further tested the priming capability of Spd and its different concentrations toward activin-induced DE differentiation. We used Spd (200 nM) for the first day and activin A (50 ng/ml) for the following three days (Spd-A50) and after that, we attempted to replace activin A with IDE1/2. Our study showed that treatment of hESCs with Spd-A50 lead to endodermal differentiation. However activin A could not be replaced by SM IDE1/2.

RNA isolation and real time RT-PCR
Total RNA from samples of the hES, DE, PP, PE cells and hepatocytes were extracted using an RNeasy mini kit (Qiagen, Germany, 74104) according to the manufacturer's protocol. RNA samples were treated with the RNase-free DNase Set (Qiagen, Germany, 79254) to remove contaminating genomic DNA. The total RNAs were reverse transcribed by the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, USA, K1632) using 0.2 µg of random hexamer primer and 1 μg of total RNA according to the manufacturer's instructions.
Real time RT-PCR was performed in a Rotor gene 6000 instrument (Corbett Life Sciences) using the following program. Stage 1:95˚C for 10 minutes, stage 2:95˚C for 10 seconds, 60˚C for 30 seconds and 72˚C for 30 seconds, for a total of 40 cycles. The PCR mix in each well included 10 μl of SYBR ® Premix Ex Taq™ II (RR081Q, Takara Bio. Inc.), 6 μl of dH 2 O, 1 μl each of the forward and reverse primers (5 pmol/µl), and 2 μl of single strand cDNA (16 ng/μl) in a final reaction volume of 20 μl. Relative gene expression was analyzed using the comparative Ct method, 2 −ΔΔCt (30). The output data from Rotor-Gene 6000 analysis software (version 1.7, Corbett Life Science) were transferred to Microsoft Excel for further analysis. For each sample, the relative expression level was calculated by normalization of target genes to GAPDH as a reference gene and then calibrated against day 0 hESCs. Samples were gathered from 3 to 5 independent biological replicates and all reactions were performed in duplicate. The primers used were designed using Perl Primer software (31). Primer sequences, expected product size and Gene Bank accession numbers are listed in table 1.

Immunoflourescent staining
For immunofluorescent staining, cells were fixed in 4% w/v paraformaldehyde (Sigma-Aldrich, USA, P6148) for 15 minutes, at 25˚C, permeabilized in 0.1% Triton X-100 for 10 minutes, at 25˚C, and blocked in 10% secondary antibody host serum in 0.5% BSA for 1 hour, at 37˚C, after which they were incubated for 24 hours with goat anti-human SOX17 antibody (R&D Systems, USA, AF1924) that was diluted 1:100 in 0.5% BSA at 4˚C. Cells were subsequently washed with PBS-Tween 20 (PBST) and incubated with diluted (1:400) rabbit anti-goat IgG-FITC antibody (Sigma-Aldrich, USA, F7367) for 1 hour at 25˚C followed by DNA staining with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA, D8417) for 3 minutes. Cells were observed under a fluorescence microscope (Olympus, BX51, Japan) and imaged with an Olympus DP72 digital camera mounted to the fluorescent microscope. For negative controls, primary antibodies were omitted and the same staining procedure was followed.

Statistical analysis
All experiments were conducted in at least three independent biological replicates. Data from real time RT-PCR were expressed as mean ± SD and the differences of the mean were statistically evaluated by SPSS software (version 16) using one-way analysis of variance (ANO-VA) followed by Tukey's HSD and LSD posthoc tests. P values less than 0.05 were considered significant.

Treating Royan H6 hESCs with Spd prior to activin induction
Suppression of regulators of a pluripotency state can lead to the priming of ESCs for differentiation into different lineages (26,27,32). Treating hESCs with Spd has been shown to lead to the down regulation of c-MYC which is known as a main factor in self-renewal of hESCs. As shown in figure 1A, we treated Royan H6 hESCs with 200 nM Spd for one day followed by 50 ng/ml activin A for the next three days (Spd-A50). We used a previously reported protocol (W/A100-A100) (11) as the positive control and DMSO treatment as the negative control.
To evaluate the efficiency of our experimental groups for endoderm induction, by the fourth day, we analyzed the cells for expressions of the following DE markers: SOX17, FOXA2 and CXCR4 (33,34); and SOX7 [visceral endoderm (VE) marker], SOX1 (neuroectoderm marker) and OCT4 (pluripotency marker) by Q-PCR (Fig 1B).

SOX17, FOXA2
and CXCR4 expression levels of Spd-A50-treated hESCs compared with the positive control (W/A100-A100-treated cells) revealed that the Spd-A50 regimen lead to the upregulation of DE marker genes at levels comparable to W/A100-A100. However, the levels of SOX7 and SOX1 expression remained unchanged and OCT4 down regulated during hESCs treatment with both Spd-A50 and W/ A100-A100 (Fig 1B).
To corroborate the SOX17 gene expression re-sults in Spd-A50-treated cells, we investigated SOX17 protein expression. SOX17+ populations were detected by immunofluorescent staining in the cultures treated with the Spd-A50 protocol (Fig 1C). Similar populations were also detected in the cultures treated with W/A100-A100. Therefore, Spd-A50 protocol was able to upregulate DE-specific markers in hESCs and produce DE cells.

Effects of Spd and activin concentrations on the efficiency of Spd-A50 protocol
The primary concentration of Spd (200 nM) was replaced by higher concentrations (400 and 800 nM) of Spd in the Spd-A50 protocol. Treated hESCs were investigated for the expressions of DE markers SOX17 and FOXA2 by Q-PCR. Although the levels of DE marker genes in Spd800-A50-treated hESCs were significantly (p<0.05) higher than Spd-A50-treated cells (Fig  2A), phase contrast images showed that treating hESCs with 800 nM Spd lead to a very high cell mortality ( Fig 2B). As the SOX17 and FOXA2 expression levels were not significantly different in Spd 400 and Spd-A50-treated cells, the 200 nM concentration of Spd was selected for the remainder of the experiments. Additionally, the lower concentration of activin A (25 ng/ml) was tested (Spd-A25). According to Q-PCR results, this concentration of activin A caused a significant decrease in the expression levels of DE marker genes.
The Spd-A50 protocol was tested on another hESC line (Royan H5) and the same gene expression results as hESC-RH6 were obtained (Fig 2C).

Effects of Spd and activin concentrations on the efficiency of Spd-A50 protocol
It has previously been shown that treatment of hESCs with IDE1/2 induced DE differentiation (25). Thus we replaced activin A with IDE1/2 in the Spd-A50 protocol and treated hESCs with this new regimen (Spd-IDE1/2). As shown in figure 3, this replacement caused a dramatic decrease in SOX17 (also at the protein level) and FOXA2 expression. However, the VE marker, SOX7 up regulated in cells treated with IDE1/2.

A. After treating hESCs with Spd-IDE1/2, samples were analyzed for the expressions of SOX17, FOXA2 and SOX7 by Q-PCR at the end of the fourth day. The target gene expression level was normalized to GAPDH and presented relative to hESC. Data are presented as mean ± SD. Asterisk indicates significant difference between the shown groups (p<0.05 as determined by ANOVA
with Tukey's LSD test; n=3).

Discussion
For DE differentiation, we used a twostep protocol. In the first step, hESCs were treated with Spd, a previously introduced SM suppressor of pluripotency (26,27) and in the second step they were exposed to activin A, an inducer of endoderm formation. We showed that Spd-A50 regimen lead to upregulation of DE-specific markers (SOX17, FOXA2 and CXCR4) while no increase was observed in VE-specific gene (SOX7) expression. There was no difference in the DE-specific gene expression patterns between Spd-A50-and W/A100-A100-induced DE cells. As reported before, Spd has been shown to inhibit nuclear localization of nucleoside diphosphate kinase B (DNPK B), which led to the down regulation of c-MYC a known main factor in hESC self-renewal. Thus it could prime hESCs for differentiation (26). As we previously reported (27), pre-treatment of hESCs with SM suppressors of pluripotency could be a strategy to improve the efficiency of DE induction.
Activin/Smad2/3 is an important signaling pathway during endoderm formation (15,35) and widely used in DE differentiation protocols. We reduced the concentration of activin A to 25 ng/ml in the Spd-A50 protocol, which led to a dramatic reduction in the expression of DE-specific genes. This agreed with previous reports that different concentrations of growth factor activin A have different inducing effects on hESCs (3). IDE1 and IDE2 have been previously reported to be inducers of DE. While the exact mechanisms of their action are not known, the activation of the TGF-β signaling pathway has been shown in hESCs treated with these SMs (25). In contrast, we did not observe any up regulation of DE markers in Spd-IDE1/2-treated hESCs while the VE marker gene (SOX7) showed a significant up regulation in these cells. This was possibly due to the different abilities of hESC lines for differentiation under identical conditions as reported in numerous studies (36).

Conclusion
This study showed that while the Spd-A50 protocol could lead to efficient DE differentiation, replacement of activin A with two previously reported inducers of DE (IDE1/2) dramatically reduced the efficiency of DE differentiation in the tested hESC lines.